5.6 The nucleolus and nuclear import and export

The messenger RNA is not the only RNA family for which the nucleus is
responsible. Ribosomal RNA transcription and processing, and
ribosome assembly are crucial aspects of nuclear function. Indeed the
site of these activities is the most prominent morphological feature within the
nucleus; it’s been known for many years as a body called the nucleolus.
Genes specifying ribosomal sequences are transcribed into pre-ribosomal-RNAs
(pre-rRNAs). Virtually all cellular RNAs undergo post-transcriptional processing
and modification. Pseudouridylation (which is the conversion of uridine to
pseudouridine at specific sites in an RNA sequence) and 2’-O-methylation of the
ribose sugar are the most common internal modifications. They are applied to
three major types of stable RNA: the spliceosomal snRNAs, ribosomal RNAs (rRNAs)
and transfer RNAs (tRNAs). As is the case for pre-mRNA processing, pre-rRNA
processing is done by families of proteins working with numerous small
nucleolar RNAs (snoRNAs) in the form of RNP
complexes (snoRNPs) that base pair with specific parts
of the pre-rRNA sequence to carry out their modification.

The snoRNAs are themselves produced from precursor transcripts and have to be
targeted to the nucleolus so that they can guide the critical modifications of
the rRNA. As we’ve indicated above, the snoRNPs can also modify spliceosomal
snRNAs and as this could affect the way pre-mRNAs are processed it is evident
that the nucleolus is responsible for more than just ribosome
assembly. Nevertheless, the nucleolus
is where ribosomal subunits are manufactured and the
proteins needed to assemble ribosomes are synthesised in the cytoplasm, like all
proteins, so they must be imported into the nucleus and also targeted to the
nucleolus. Subsequently, ribosomal subunits need to be re-exported to the
cytoplasm. Ribosomal subunits are large macromolecular assemblies and the way
they are translocated to the cytoplasm is not yet known. More is known about
export of mRNA from the nucleus.

The nucleus is separated from the cytoplasm by a double membrane called the
nuclear envelope, which isolates and protects the DNA from the turmoil of the
cytoplasm (in prokaryotes the processes that depend on DNA take place in the
cytoplasm). In eukaryotes many proteins and RNAs are transported across the
nuclear envelope. This is known as nuclear–cytoplasmic trafficking
and features both import into and export from the nucleus. It
occurs through the nuclear pore complexes (NPCs),
which are complex assemblies that are embedded in the double-membrane of the
nuclear envelope.
The NPCs provide channels (about 9 nm in diameter)
that allow passage of ions and small molecules (less than about 50 kDa) by
diffusion, but proteins, RNAs, and ribonucleoprotein (RNP) particles larger than
9 nm are selectively transported through NPCs by an energy-dependent mechanism.
The trafficking is selectively regulated by developmental and environmental
signals.

The overall three-dimensional architecture of the NPC is conserved from yeast
to higher eukaryotes though the yeast NPC is smaller than those found in
vertebrates. The protein components of NPCs are called nucleoporins;
the yeast NPC is composed of 30-50 different nucleoporins (up to 100 in
vertebrates). We are just beginning to understand the three-dimensional
molecular architecture of the NPC but, crudely, it looks rather like the ball
valve of a swimmer’s snorkel. There is a ring of nucleoporins on the cytoplasmic
side of the pore that extends through the nuclear membrane, matched with another
ring on the nuclear side of the pore. Between the rings there is a central plug
(also called the transporter
because some specimens seem to have cargo trapped within), and eight short
fibrils extend from the cytoplasmic ring into the cytoplasm whereas the nuclear
ring anchors a basket made from eight long thin filaments (Upla et al.,
2017; Kosinski et al., 2016).

How this highly organised tunnel actually works remains a mystery, but the
transport process itself depends on a family of soluble transporter
proteins known as importins or exportins
(both also called karyopherins) that carry proteins and RNA
between the cytoplasm and nucleus. Proteins or RNAs ready for transport (the
cargo) contain specific sequences that identify them. The nuclear
localisation signal (NLS) is recognised by importins,
and the nuclear export signal (NES) sequence
is recognised by exportins. Transporter and cargo then dock at the mouth of the
NPC and deliver the transport cargo. Importantly, for large RNA molecules such
as mRNA - which is translocated as a RNP - the signals for export are not the
mRNA itself but on the proteins, mostly hnRNP and splicing factors, that have
guided the transcript through final processing. This emphasises again the
integration and co-operation of the machineries that function in gene expression
(Wente & Rout, 2010; Marfori et al., 2011).

Interaction of cargo with transporter is modulated by the small protein Ran,
which is a GTPase. GTP hydrolysis by Ran does not provide the energy for
transport but regulates the assembly and disassembly of transport
complexes. Effectively, the nucleotide bound state of Ran identifies
the compartment: Ran-GTP in the nucleus, Ran-GDP in the cytoplasm. For
importins, the binding of cargo and Ran-GTP is antagonistic (so importins drop
their cargo in the nucleus) and for exportins the binding of cargo and Ran-GTP
is co-operative (so exportins are loaded in the nucleus). Of course, it’s not as
simple as that; there are several accessory factors involved in maintaining Ran
in its proper GTP or GDP bound state, including a nuclear nucleotide exchange
factor (RCC1), a cytoplasmic Ran-GTPase activating protein (RanGAP), a protein
called RanBP1, and nuclear transport factor 2 (NTF2). Though the significance of
this is uncertain, there are multiple signals and multiple pathways for both
nuclear import and export; 14 members of the importin/exportin family are known
in yeast and about 19 in humans (Chook & Süel, 2011).

By this stage in our description we’ve reached the point of exporting the
gene transcript(s) to the cytoplasm. We’ll continue our story of what
subsequently happens to the ribosomal subunits and mRNAs in the cytoplasm later
[it's in Section 5.10;
CLICK HERE to see it now if you just can't wait]. At the moment we want to
proceed to the other prime function of the nucleus, namely storage and
transmission of the cell’s genetic content.